Nucleotide-Dependent Interaction of Saccharomyces cerevisiae Hsp90 with the Cochaperone Proteins Sti1, Cpr6, and Sba1

Effect of mutations predicted to affect lid closure and N-terminal dimerization. A. Cell extracts were prepared from yeast expressing His-Hsc82 WT or His-Hsc82-A107N. His-Hsc82 complexes were isolated from lysates incubated for 5 min at 30°C in the presence of no exogenous nucleotide, 5 mM AMP-PNP, or 5 mM ATP plus an ATP-regenerating system as indicated. Nickel-bound protein complexes were separated by SDS-PAGE and analyzed as described in the legend to Fig. 1. B. As above, except that WT His-Hsc82, His-Hsc82-T22I, or His-T101I was isolated from cells coexpressing WT untagged Hsp82, since hsc82-T22I and hsc82-T101I confer a lethal phenotype.

Effect of alteration of residues in the catalytic loop. Cell lysates from strains expressing indicated His-Hsc82 mutants were isolated, supplemented with a nucleotide, and incubated as described for Fig. 1, except that the effect of 5 mM ADP was also monitored. His-Hsc82 mutants unable to support viability of an hsc82 hsp82 strain (E33A, R376A, and E377K mutants) were coexpressed along with untagged WT Hsp82. In the remaining cases (WT and N373A and Q380A mutants), His-Hsc82 was the only Hsp90 protein expressed in the cell. Nickel-bound protein complexes were separated by SDS-PAGE. Upper panels, His-Hsc82 present in a Coomassie blue-strained gel. Lower panels, immunoblots using antibodies specific for Sti1, Cpr6, or Sba1.

Effect of additional Hsc82 mutations on cochaperone interaction in the presence of AMP-PNP. WT and mutant His-Hsc82 complexes were isolated and analyzed as described in the legend to Fig. 3 except that lysate was supplemented with 5 mM AMP-PNP. In all cases, WT or mutant His-Hsc82 was the only Hsp90 protein present in the cell.

Interaction of mutant Hsc82 with Sti1, Sba1, and Cpr6 in the presence of ATP plus an ATP-regenerating system. His-Hsc82 complexes were isolated and analyzed as for Fig. 4, except that samples were supplemented with 5 mM ATP+RS. In all cases, WT or mutant His-Hsc82 was the only Hsp90 protein present in the cell.

Cpr6 interacts with His-Hsc82 in a strain lacking Sba1. Untagged Hsc82 (lanes 1 to 3) or His-Hsc82 (lanes 4 to 6) was expressed in strain JJ816 (hsc82 hsp82). In lanes 7 to 9, His-Hsc82 was expressed in strain JJ40 (hsc82 hsp82 sba1). Cell lysates were isolated and supplemented with a nucleotide as described in the legend to Fig. 1: lanes 1, 4, and 7, no exogenous nucleotide; lanes 2, 5 and 8, 5 mM AMP-PNP; lanes 3, 6, and 9, 5 mM ATP plus an ATP regenerating system. For the upper panel, nickel resin-bound protein complexes were separated by SDS-PAGE (7.5% acrylamide) followed by staining with Coomassie blue. For the lower panels, protein complexes were subjected to SDS-PAGE followed by immunoblot analysis using antibodies against the indicated proteins. In the panel marked “lysate,” whole-cell extract was separated by SDS-PAGE and immunoblotted with an antibody specific for Sba1 to confirm the lack of expression of Sba1 in the hsc82 hsp82 sba1 strain.